The mannose binding lectin Griffithsin (GRFT) represents an exciting new approach to preventing infection by HIV-1 and herpes simplex virus 2 (HSV-2). GRFT binds the dense glycan shield on the HIV-1 and HSV-2 envelopes, for which oligomannose structures are a highly conserved feature. In protecting against HIV, GRFT possesses potent neutralizing activity towards diverse viral strains, and it acts synergistically with chemical antimicrobials and even broadly neutralizing antibodies. In protecting against HSV-2, GRFT has been shown to prevent infection and stop cell-to-cell spread both in vitro and in pre-clinical animal models. Importantly, HSV-2 infection increases the risk of HIV-1 acquisition by 3- to 5-fold in the general population and at least doubles the HIV-1 risk in high risk populations. Thus, GRFT?s capacity to protect against infection by both viruses suggests it is a promising agent for Pre-exposure Prophylaxis (PrEP), and we propose that a GRFT-based PrEP microbicide could help dramatically reduce new cases of HIV-1 infection. Indeed, under the NIAID- funded PREVENT Program, we recently received FDA approval for a first-in-human study of our Q-GRFT topical microbicide (Q-GRFT is a variant engineered for enhanced oxidative stability). Notably, as part of GLP toxicology studies for this Investigational New Drug (IND) application, we observed a potent anti-drug antibody (ADA) response in rabbits. This prompted a closer examination of Q-GRFT immunogenicity in two clinically relevant models: human peripheral blood mononuclear cell (PBMC) immunoassays and humanized HLA transgenic mice. We found that Q-GRFT elicited high ADA titers following repeated dosing in HLA transgenic mice, and it was similarly shown to activate helper T cells in PBMC from several healthy human donors. Thus, while Q-GRFT holds great promise in the fight against new HIV-1 infections, our preclinical data strongly suggests that the immunogenicity issue must be addressed if Q-GRFT is to be employed, long-term, as a PrEP agent. We propose here to design and develop a next-generation deimmunized GRFT (dGRFT) microbicide that retains the potent and broad-spectrum antiviral activity of wild type and Q-GRFT but evades detrimental anti-drug immune responses in humans. To achieve this goal, we will combine Stealth Biologics? advanced protein design and deimmunization platform with the deep expertise and experience of the development team, which has already pushed Q-GRFT into clinical trials. In Phase I of this Fast-track SBIR proposal, we will develop a dGRFT lead candidate and validate it using preliminary immunological and functional assays. In Phase II of this Fast-track proposal, we will confirm dGRFT?s breadth of antiviral activity, demonstrate reduced immunogenic potential in large and diverse PBMC donor panels, develop a scalable manufacturing system, assess dGRFT toxicity in both standard and humanized animal models, and test the agent?s prophylactic efficacy in clinically-relevant animal models of HSV-2 and HIV infection. Upon completion of this grant, we will have in hand a high-performance dGRFT lead candidate that is positioned for IND-enabling studies.